The objective of this project is to Genotype
three samples (CONTROL Versus TWO TREATED Varieties) of the
same line with five primers on Cashew leaves of V4 variety.
Job Requirement:
Isolation of genomic DNA from Cashew
leaves samples of V4 variety.
Generating RAPD-Fingerprinting profiles
showing Polymorphic bands with five RAPD primers
MATERIALS & METHODS:
Control & Two Treated Cashew leaves were provided by
Mr. Mahendra Kumar Trivedi on 09.03.2008.
Genomic DNA was isolated using Nucleon Phytopure Plant DNA Extraction
Kit (Amersham Bioscience: Cat# RPN 8510)
Steps Involved:
Genomic DNA was isolated from Cashew
leaves using Nucleon Phytopure Plant DNA Extraction Kit
(Amersham Bioscience: Cat# RPN 8510)
Performed RAPD reactions using five RAPD
primers (RPL Series used separately) to find polymorphism
between control and two treated leaves under PCR conditions
as mentioned:
Cycle condition:
94°C
94°C
35°C
72°C
94°C
38°C
72°C
72°C
4 min
60 sec
60 sec
120 sec
60 sec
60 sec
90 sec
7 min
10 cycles
35 cycles
12 of PCR products were loaded on to 1.5 % agarose gel and
resolved by electrophoresis. The gel was subsequently stained
with ethidium bromide and viewed under UV-light. Photographs
were documented subsequently (attached).
Polymorphic DNA bands are marked by arrows on the copy of the report.
Fig. 1. RAPD Profile of Cashew samples Generated
using Genei RAPD primer
RPL 4A, RPL 13A, RPL 18A, RPL 19A & RPL 20A.
Lane Description:
C1: Control leaves
A1: Treated leaves
G1: Treated leaves
M:100 bp Ladder (GeneiTM) Cat # RMBD19S;
Calculation:
A/ Bx100 = Percent polymorphism
Where,
A= Polymorphic bands in
treated plant
B= No of bands in control
Sl.
No
Bands scored
Description
Primer
1
12
Two bands are Common to Control and
Treated samples; Two bands are unique to Control sample;
One band is unique to Treated sample A1; Two bands are
unique to Treated sample G1.
RPL 4A
2
13
Four bands are Common to Control and
Treated samples; One band is unique to Control sample;
Two bands are unique to Treated sample A1; Four bands
are unique to Treated sample G1.
RPL 13A
3
12
Eight bands are Common to Control and
Treated samples; No band is unique to Control sample;
No band is unique to Treated sample A1; Three bands
are unique to Treated sample G1.
RPL 18A
4
10
Five bands are Common to Control and
Treated samples; No band is unique to Control sample;
Two bands are unique to Treated sample A1; Five bands
are unique to Treated sample G1.
RPL 19A
5
Not worked
RPL 20A
RESULTS & DISCUSSION:
1. The level of true polymorphism between Control & Treated
A1 was evaluated at 42 % with RPL 4A primer.
2. The level of true polymorphism between Control & Treated
A1 was evaluated at 60 % with RPL 13A primer.
3. The level of true polymorphism between Control & Treated
A1 was evaluated at 0 % with RPL 18A primer.
4. The level of true polymorphism between Control & Treated
A1 was evaluated at 0 % with RPL 19A primer.
5. The level of true polymorphism between Control & Treated
G1 was evaluated at 42 % with RPL 4A primer.
6. The level of true polymorphism between Control & Treated
G1 was evaluated at 100 % with RPL 13A primer.
7. The level of true polymorphism between Control & Treated
G1 was evaluated at 37 % with RPL 18A primer.
8. The level of true polymorphism between Control & Treated
G1 was evaluated at 100 % with RPL 19A primer.
CONCLUSION:
Polymorphism was detected between Control & Treated
(A1) samples. The percentage of true polymorphism observed
between Control & Treated sample A1 of Cashew V4 variety
is on an average 20%.
Polymorphism was detected between Control & Treated
(G1) samples. The percentage of true polymorphism observed
between Control & Treated sample G1 of Cashew V4 variety
is on an average 55%.
The treatment modality of Mr. Mahendra Kumar Trivedi is
not known to us, the company is only concerned about detecting
the DNA Polymorphism in Control & Two Treated lines
of Cashew leaves varieties.
of PCR products were loaded on to 1.5 % agarose gel and
resolved by electrophoresis. The gel was subsequently stained
with ethidium bromide and viewed under UV-light. Photographs
were documented subsequently (attached).
on the copy of the report.
